The invention relates to vaccines that include recombinant flaviviruses.
Flaviviruses are small, enveloped, positive-strand RNA viruses that are generally transmitted by infected mosquitoes and ticks. Several flaviviruses, such as yellow fever, dengue, Japanese encephalitis, tick-borne encephalitis, and West Nile viruses, pose current or potential threats to global public health. Yellow fever virus, for example, has been the cause of epidemics in certain jungle locations of sub-Saharan Africa, as well as in some parts of South America. Although many yellow fever virus infections are mild, the disease can also cause severe, life-threatening illness. The initial or acute phase of the disease state is normally characterized by high fever, chills, headache, backache, muscle ache, loss of appetite, nausea, and vomiting. After three to four days, these symptoms disappear. In some patients, symptoms then reappear, as the disease enters its so-called toxic phase. During this phase, high fever reappears and can lead to shock, bleeding (e.g., bleeding from the mouth, nose, eyes, and/or stomach), kidney failure, and liver failure. Indeed, liver failure causes jaundice, which is yellowing of the skin and the whites of the eyes, and thus gives “yellow fever” its name. About half of the patients who enter the toxic phase die within 10 to 14 days. However, persons that recover from yellow fever have lifelong immunity against reinfection. The number of people infected with yellow fever virus over the last two decades has been increasing, with there now being about 200,000 yellow fever cases, and about 30,000 associated deaths, each year. The re-emergence of yellow fever virus thus presents a serious public health concern.
West Nile (WN) virus has a wide distribution in Africa, the Indian subcontinent, Europe, Ukraine, Russia, Central Asia, and the Middle East (Monath and Heinz, in Virology 3rd ed., Fields et al., eds., Lippincott-Raven, pp. 961-1034, 1995). In 1999, an unprecedented epidemic of encephalitis in humans and horses caused by WN virus occurred in the United States (Enserik, Science 286:1450-1451, 1999). Since then, the virus has become permanently established in the Americas, affecting nearly the entire territory of the U.S. Thus far the record year in terms of morbidity/mortality in the U.S. was 2003, with 9862 reported cases, of which approximately one-third were accompanied by neurological symptoms, and 264 deaths. The human disease varies from mild dengue-like illness to fatal meningoencephalitis, with the most severe illness occurring in the elderly. To date, there is no effective drug treatment against West Nile virus and methods of surveillance and prevention are not significantly impacting the number of cases of human infection. The risks of the virus migrating into South America, as well as epidemics in underdeveloped countries, are extremely high. The development of a safe and effective vaccine will contribute to the control of future epidemics.
Flaviviruses, including yellow fever virus and West Nile virus, have two principal biological properties responsible for their induction of disease states in humans and animals. The first of these two properties is neurotropism, which is the propensity of the virus to invade and infect nervous tissue of the host. Neurotropic flavivirus infection can result in inflammation of and injury to the brain and spinal cord (i.e., encephalitis), impaired consciousness, paralysis, and convulsions. The second of these biological properties of flaviviruses is viscerotropism, which is the propensity of the virus to invade and infect vital visceral organs, including the liver, kidney, and heart. Viscerotropic flavivirus infection can result in inflammation and injury of the liver (hepatitis), kidney (nephritis), and cardiac muscle (myocarditis), leading to failure or dysfunction of these organs.
Neurotropism and viscerotropism appear to be distinct and separate properties of flaviviruses. Some flaviviruses are primarily neurotropic (such as West Nile virus), others are primarily viscerotropic (e.g., yellow fever virus and dengue virus), and still others exhibit both properties (such as Kyasanur Forest disease virus). However, both neurotropism and viscerotropism are present to some degree in all flaviviruses. Within a host, an interaction between viscerotropism and neurotropism is likely to occur, because infection of viscera occurs before invasion of the central nervous system. Thus, neurotropism depends on the ability of the virus to replicate in extraneural organs (viscera). This extraneural replication produces viremia, which in turn is responsible for invasion of the brain and spinal cord.
Fully processed, mature virions of flaviviruses contain three structural proteins, capsid (C), membrane (M), and envelope (E). Seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) are produced in infected cells. Both viral receptor binding and fusion domains reside within the E protein. Further, the E protein is also a desirable component of flavivirus vaccines, since antibodies against this protein can neutralize virus infectivity and confer protection on the host against the disease. Immature flavivirions found in infected cells contain pre-membrane (prM) protein, which is a precursor to the M protein. The flavivirus proteins are produced by translation of a single, long open reading frame to generate a polyprotein, followed by a complex series of post-translational proteolytic cleavages of the polyprotein, to generate mature viral proteins (Amberg et al., J. Virol. 73:8083-8094, 1999; Rice, “Flaviviridae,” In Virology, Fields et al., ed., Raven-Lippincott, New York, Volume I, p. 937, 1995). The virus structural proteins are arranged in the N-terminal region of the polyprotein in the order C-prM-E, while the non-structural proteins are located in the C-terminal region, in the order noted above.
Live vaccines confer the most potent and durable, protective immune responses against disease caused by viral infections. In the case of flaviviruses, the development of a successful vaccine requires that the virulence properties are modified, so that the vaccine virus has reduced neurotropism and viscerotropism for humans or animals. Several different approaches have been used in the development of vaccines against flaviviruses. In the case of yellow fever virus, two vaccines (yellow fever 17D and the French neurotropic vaccine) have been developed by serial passage (Monath, “Yellow Fever,” In Plotkin and Orenstein, Vaccines, 3rd ed., Saunders, Philadelphia, pp. 815-879, 1999). The yellow fever 17D vaccine was developed by serial passage in chicken embryo tissue, and resulted in a virus with significantly reduced neurotropism and viscerotropism. The French neurotropic vaccine was developed by serial passages of the virus in mouse brain tissue, and resulted in loss of viscerotropism, but retained neurotropism. Indeed, a high incidence of neurological accidents (post-vaccinal encephalitis) was associated with the use of the French vaccine.
Another approach to attenuation involves the construction of chimeric flaviviruses, which include components of two (or more) different flaviviruses. Chimeric flaviviruses have been made that include structural and non-structural proteins from different flaviviruses. For example, the so-called ChimeriVax™ technology employs the yellow fever 17D virus capsid and nonstructural proteins to deliver the envelope proteins (prM and E) of other flaviviruses (see, e.g., Chambers et al., J. Virol. 73:3095-3101, 1999). Indeed, this technology has been used to develop vaccine candidates against dengue viruses, Japanese encephalitis (JE) virus, West Nile virus, and St. Louis encephalitis (SLE) virus (see, e.g., Pugachev et al., in New Generation Vaccines, 3rd ed., Levine et al., eds., Marcel Dekker, New York, Basel, pp. 559-571, 2004; Chambers et al., J. Virol. 73:3095-3101, 1999; Guirakhoo et al., Virology 257:363-372, 1999; Monath et al., Vaccine 17:1869-1882, 1999; Guirakhoo et al., J. Virol. 74:5477-5485, 2000; Arroyo et al., Trends Mol. Med. 7:350-354, 2001; Guirakhoo et al., J. Virol. 78:4761-4775, 2004; Guirakhoo et al., J. Virol. 78:9998-10008, 2004; Monath et al., J. Infect. Dis. 188:1213-1230, 2003; Arroyo et al., J. Virol. 78:12497-12507, 2004; and Pugachev et al., Am. J. Trop. Med. Hyg. 71:639-645, 2004). These are live viral vaccines, which, similar to the YF17D vaccine, elicit strong humoral and cellular immune responses directed against a desired heterologous virus. Based on extensive characterization of ChimeriVax™-JE and dengue vaccines, the main features of ChimeriVax™ vaccines have been observed to include the ability to replicate to high titers in substrate cells (7 log10 pfu/ml or higher), low neurovirulence in weanling and infant mice (significantly lower compared to YF17D), high attenuation in formal monkey tests for neurovirulence and viscerotropism, high genetic and phenotypic stability in vitro and in vivo, inefficient replication in mosquitoes, which is important to prevent uncontrolled spread in nature, and the induction of robust protective immunity in mice, monkeys, and humans following administration of a single dose, without serious post-immunization side effects.
In other approaches to attenuation, mutagenesis of flaviviruses, including chimeric flaviviruses, has been undertaken. Several experimental approaches to attenuation of wild type flavivirus pathogens have been described (see, e.g., reviewed by Pugachev et al., Int. J. Parasitol. 33:567-582, 2003). For example, it has been found that mutations in certain amino acids of the envelope proteins of chimeric flaviviruses including capsid and non-structural proteins of yellow fever virus and membrane and envelope proteins of Japanese encephalitis virus, a dengue virus, or West Nile virus decrease viscerotropism (see, e.g., WO 03/103571 and WO 2004/045529). Another approach, originally applied to wild type dengue-4 virus, involves large deletions of 30 nucleotides or more in the 3′ untranslated region (3′UTR; Men et al., J. Virol. 70:3930-3937, 1996; U.S. Pat. No. 6,184,024 B1). One of these deletions, named deletion delta 30 or Δ30, was further studied in the context of wild type dengue-4 and dengue-1 viruses and a dengue-4/WN chimeric virus (Durbin et al., AJTMH 65:405-413, 2001; Whitehead et al., J. Virol. 77:1653-1657, 2003; Pletnev et al., Virology 314:190-195, 2003; WO 03/059384; WO 03/092592; WO 02/095075). Additionally, some of the large 3′UTR deletions (417-616 nucleotides long) introduced into wild type tick-borne encephalitis (TBE) and Langat viruses were found to be highly attenuating in a mouse model (Mandl et al., J. Virol. 72:2132-2140, 1998; Pletnev, Virology 282:288-300, 2001). A limited amount of in vitro data was published for YF17D vaccine virus. Specifically, Bredenbeek and co-authors demonstrated that a large deletion of all three repeat sequence (RS) elements of the 3′UTR (188 nucleotides in length; the location of the RS elements is illustrated in FIG. 1A) or a 25 nucleotide deletion of the conserved sequence element 2 (CS2) did not preclude virus replication in BHK cells, while three other deletions (25-68 nucleotides in length) that affected CS1 or the 3′ extreme stem-and-loop were lethal (Bredenbeek et al., J. Gen. Virol. 84:1261-1268, 2003). Others have shown that mutations introduced into the large 3′ terminal stem-loop structure of the flavivirus (dengue) 3′UTR resulted in attenuation, while retaining the ability of the virus to immunize the host (Markoff et al., J. Virol. 76:3318-3328, 2002).
A second approach that was described for attenuation of a highly pathogenic wild type TBE virus utilized relatively large deletions in the capsid protein C, as described by Kofler and co-workers, who introduced a series of deletions into the C protein of TBE virus and recovered several viable mutants (Kofler et al., J. Virol. 76:3534-3543, 2002). Specifically, a 16-amino acid deletion in the central hydrophobic domain of the protein (predicted Helix I; see in FIG. 2A) drastically reduced virus replication in BHK cells and significantly decreased neuroinvasiveness in mice. Immunization with this TBE mutant protected mice from challenge with highly pathogenic TBE strain Hypr (>100 LD50) (Kofler et al., J. Virol. 76:3534-3543, 2002).
Approved vaccines are not currently available for many medically important flaviviruses having viscerotropic properties, such as West Nile, dengue, and Omsk hemorrhagic fever viruses, among others.